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. 2012 Jun 25;590(Pt 17):4391–4400. doi: 10.1113/jphysiol.2012.235952

The hyperaemic response to passive leg movement is dependent on nitric oxide: a new tool to evaluate endothelial nitric oxide function

Stefan P Mortensen 1, Christopher D Askew 2,3, Meegan Walker 3, Michael Nyberg 4, Ylva Hellsten 4
PMCID: PMC3473293  PMID: 22733658

Abstract

Passive leg movement is associated with a ∼3-fold increase in blood flow to the leg but the underlying mechanisms remain unknown. The objective of the present study was to examine the role of nitric oxide (NO) for the hyperaemia observed during passive leg movement. Leg haemodynamics and metabolites of NO production (nitrite and nitrate; NOx) were measured in plasma and muscle interstitial fluid at rest and during passive leg movement with and without inhibition of NO formation in healthy young males. The hyperaemic response to passive leg movement and to ACh was also assessed in elderly subjects and patients with peripheral artery disease. Passive leg movement (60 r.p.m.) increased leg blood flow from 0.3 ± 0.1 to 0.9 ± 0.1 litre min−1 at 20 s and 0.5 ± 0.1 litre min−1 at 3 min (P < 0.05). Mean arterial pressure remained unchanged during the trial. When passive leg movement was performed during inhibition of NO formation (NG-mono-methyl-l-arginine; 29–52 mg min−1), leg blood flow and vascular conductance were increased after 20 s (P < 0.05) and then returned to baseline levels, despite an increase in arterial pressure (P < 0.05). Passive leg movement increased the femoral venous NOx levels from 35 ± 5 at baseline to 62 ± 11 μmol l−1 during passive leg movement (P < 0.05), whereas muscle interstitial NOx levels remained unchanged. The hyperaemic response to passive leg movement were correlated with the vasodilatation induced by ACh (r2 = 0.704, P < 0.001) and with age (r2 = 0.612, P < 0.001). Leg blood flow did not increase during passive leg movement in individuals with peripheral arterial disease. These results suggest that the hypaeremia induced by passive leg movement is NO dependent and that the source of NO is likely to be the endothelium. Passive leg movement could therefore be used as a non-invasive tool to evaluate NO dependent endothelial function of the lower limb.

Key points

  • Passive leg movement is associated with a ∼3-fold increase in blood flow to the leg, but the underlying mechanisms remain unknown.

  • Passive leg movement increased venous levels of metabolites of nitric oxide (NO) in young subjects, whereas they remained unaltered in the muscle interstitial space. Inhibition of NO synthesis lowered the vasodilatory response to passive leg movement by ∼90%.

  • The increase in leg blood flow was lower in elderly subjects compared to young subjects and leg blood flow did not increase when passive leg movement was performed by elderly with peripheral artery disease.

  • The results suggest that the hyperaemia induced by passive leg movement is NO dependent. The hyperaemic response to passive leg movement and to ACh was also assessed in elderly subjects and patients with peripheral artery disease.

Introduction

Endothelial dysfunction is associated with a reduced formation of endothelium-dependent vasodilators including nitric oxide (NO) (Widlansky et al. 2003) and plays a key role in the pathogenesis of micro- and macrovascular complications observed in pathological conditions such as diabetes, atherosclerosis, hypertension and peripheral artery disease (Tooke, 1995; Taddei et al. 1997; Ross, 1999; Tendera et al. 2011). Evaluation of endothelial function and especially NO function is an important clinical tool, but the currently available methods for quantification of endothelial function are invasive or induce vasodilatation not only by NO-dependent pathways (Widlansky et al. 2003; Tschakovsky & Pyke, 2005).

Passive leg movement increases limb blood flow (Rådegran & Saltin, 1998; Krustrup et al. 2004; Wray et al. 2005), with no increase in muscle activity (Hellsten et al. 2008) and little (González-Alonso et al. 2008; Høier et al. 2010) or no (Krustrup et al. 2004; Hellsten et al. 2008) increase in metabolism. Furthermore, the increase in leg blood flow occurs independently of the arousal invoked by passive movement or the thought of passive leg movement (Venturelli et al. 2011). Mechanical factors are therefore likely to be involved in the increase in blood flow during passive leg movement, but to what extent locally formed vasodilating compounds mediate the increase in flow remains unknown. One study has suggested NOS inhibition has no effect on blood flow during passive movement, but in this study only five to seven passive leg movements were performed to accelerate the leg to 60 r.p.m. (Rådegran & Saltin, 1999). In vitro studies have demonstrated that shear stress increases the formation of NO (Pohl et al. 1986) and in vivo studies have shown that flow-mediated dilatation is partly mediated by NO (Joannides et al. 1995; Kooijman et al. 2008) and that eNOS expression is upregulated after a period of passive leg movement training (Hellsten et al. 2008; Høier et al. 2010). Both increased shear stress and stretch are inherent to passive leg movement (Høier et al. 2010) and NO is therefore a likely candidate for the mediation of passive flow. Peripheral arterial disease is characterized by a reduced endothelial NO function (Böger et al. 1997) and these patients are therefore likely to have a reduced hyperaemic response, if passive leg movement increases NO formation.

The purpose of the present study was to examine the role of NO in the hyperaemic response to passive limb movement and to determine if blood flow is lower during passive leg movement in the elderly and patients with peripheral artery disease. To accomplish this, we measured leg haemodynamics during passive leg movement with and without inhibition of NO formation and determined metabolites of NO synthesis in venous plasma and muscle interstitial fluid at rest and during passive leg movement and compared the hyperaemic response to elderly and patients with peripheral artery disease. To avoid possible confounding factors during blood sampling, we used an intravascular microdialysis probe to separate NO metabolites from blood in vivo. We hypothesized that passive leg movement results in vasodilatation via NO formation such that inhibition of NO formation would lower the hyperaemic response to passive leg movement.

Methods

Subjects

A total of 38 subjects participated in four studies (Table 1). All of the healthy young and elderly subjects were recreationally active and were not receiving any medication. Subjects with peripheral artery disease had limiting intermittent claudication and a resting ankle to brachial blood pressure index <0.9 in the affected limbs. The subjects were informed of any risks and discomforts associated with the experiments before giving their informed oral and written consent to participate. The study was approved by the Ethical committee of the Capitol Region of Denmark and the Human Research Ethics Committee of the University of the Sunshine Coast and all of the procedures followed were in accordance with institutional guidelines. The study conformed to the Declaration of Helsinki.

Table 1.

Subject characteristics

Young Middle aged Elderly PAD
Subjects (n) 15 4 11 8
Men/women 15/0 3/1 11/0 6/2
Age (years) 24 ± 5 46 ± 4 65 ± 1 68 ± 2
Weight (kg) 79 ± 8 75 ± 7 82 ± 2 83 ± 2
Height (cm) 182 ± 5 173 ± 4 176 ± 2 177 ± 2
Inline graphic (ml min−1 kg−1) 48 ± 7 33 ± 4 34 ± 2 −
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PAD: peripheral artery disease.

Experimental protocols

Study 1: Effect of NOS inhibition on the hyperaemic response to passive leg movement in young subjects

One week prior to the experimental day, the subjects visited the laboratory to become accustomed to the one-leg knee-extensor model. The subjects refrained from caffeine, alcohol and exercise for 24 h before the study. On the day of the experiment, the subjects were instructed to ingest a light breakfast 1 h before reporting to the laboratory at 08.00 h.

Catheters were placed into the femoral artery of the non-experimental leg (blood sampling and blood pressure measurement) and into the femoral artery (drug infusion) and vein (blood sampling) of the experimental leg under local anaesthesia (lidocaine). Following 30 min of supine rest, the subjects (n = 7) completed 3 min of passive leg movement under the following conditions: (1) control and (2) NG-mono-methyl-l-arginine (l-NMMA; nitric oxide synthase (NOS) inhibitor). During passive leg movement, the subjects were in an upright seated position and the lower leg was strapped to the ergometer and was moved passively at a frequency of 60 r.p.m. Saline (control) or l-NMMA (4.0 mg min−1 (kg leg mass)−1; Clinalfa, Bachem, Weil am Rhein, Germany) was infused into the femoral artery of the experimental leg for 4 min prior to passive leg movement and during the 3 min of passive leg movement. A similar dose of l-NMMA has been found to lower ACh induced increase in leg blood flow by ∼65% (Mortensen et al. 2009b). Blood samples (1–5 ml) were drawn simultaneously from the femoral artery and vein at rest and during passive leg movement (30, 60, 90, 120, 150 and 180 s). The two trials were separated by a 30 min rest period. Due to the potential long lasting effects of l-NMMA, the control trial was always performed first. To test the effect of time and reproducibility of the hyperaemic response, four subjects performed three trials of passive exercise (separated by one hour) on a separate day. No differences in the blood flow response to passive leg movement were observed.

Study 2: Effect of passive leg movement on plasma and muscle interstitial metabolites in young subjects

A catheter was placed in the femoral vein, 4–5 cm below the inguinal ligament and advanced 10 cm in the proximal direction. A microdialysis probe (CMA 70 bolt, CMA Microdialysis, Solna, Sweden) with a 10 mm membrane (20 kDa cut-off) was inserted into this catheter such that the membrane was placed in the vein. Three microdialysis probes (CMA63) were inserted into the vastus lateralis muscle of the experimental leg under local anethesia (lidocaine). Thirty minutes after insertion of the probes, the subjects performed 10 min of light (10 W, i.e. <20% maximal workload, WLmax) knee-extensor exercise with the purpose of minimizing the tissue response to insertion trauma. To re-establish resting conditions, the subjects rested for another 30 min. The subjects (n = 6) then completed 10 min of passive leg movement (60 r.p.m.). Microdialysate was collected for 10 min before and during the passive leg movement.

Study 3: Hyperaemic response to passive leg movement and arterial ACh infusion

Twelve healthy subjects were catheterized as in study 1. After 30 min of rest, femoral arterial blood flow and arterial blood pressure were measured before and during 3 min of seated passive leg movement and 3 min of supine femoral arterial ACh infusion (25 μg min−1 (kg leg mass)−1). The two trials were separated by 60 min of supine rest.

Study 4: Hyperaemic response to passive leg movement in healthy subjects and patients with peripheral artery disease

Femoral arterial blood flow was measured at rest and during 3 min of passive leg movement in four middle-aged individuals, eight elderly men and eight individuals diagnosed with peripheral artery disease.

Measurements

Femoral arterial blood flow (study 1, 3 and 4)

Femoral arterial blood flow was measured with an ultrasound machine equipped with a linear probe operating an imaging frequency of 7–9 MHz and Doppler frequency of 4.2–5.0 MHz (Logic E9, GE Healthcare in the healthy subjects and Mindray M5 in patients with peripheral artery disease). The site of blood velocity measurements in the common femoral artery was distal to the inguinal ligament but above the bifurcation into the superficial and profound femoral branch to avoid turbulence from the bifurcation. All recordings were obtained at the lowest possible insonation angle and always below 60 deg. The sample volume was maximized according to the width of the vessel, and kept clear of the vessel walls. A low-velocity filter (velocities <1.8 m s−1) rejected noises caused by turbulence at the vascular wall. Doppler tracings and B-mode images were recorded continuously and Doppler tracings were averaged over eight heart cycles (10 s values) whereas the area under the curve (AUC) was calculated from the continuous Doppler tracings. Vessel diameter was determined during each Doppler recording. Arterial diameter was calculated as 1/3 of the diameter during the systole and 2/3 of the diameter during the diastole.

Microdialysis procedure (study 2)

The microdialysis probes were continuously perfused with a Ringer solution (Fresenius Kabi AB, Sweden) with a high-precision syringe pump (CMA 102, CMA microdialysis, Solna, Sweden) at a rate of 5 μl min−1. The intravascular probe was also perfused with Dalteparin (25 IE ml−1; Fragmin, Pfizer Inc., USA) to avoid blood clotting in the membrane. A small amount (2.7 nm) of [2-3H]ATP (<0.1 μCi ml−1) was added to the perfusate for the calculation of probe recovery. The purpose of determining probe recovery was to correct for differences in recovery occurring in the transition from rest to passive leg movement. After collection of samples, the microdialysate was weighed, and the actual flow rate was calculated to estimate any loss of fluid or abnormal decrease in perfusion rate and the samples were then stored at −80°C for later analysis. The relative loss for each probe was determined according to the internal reference method (Scheller & Kolb, 1991; Jansson et al. 1994). The molecular probe recovery (PR) was calculated as [PR = (dpminfusate× dpmdialysate)/dpminfusate], where dpm denotes disintegrations per minute. The 3H activity (in dpm) was measured on a liquid scintillation counter (Tri-Carb 2000; Copenhagen; Denmark) after addition of the infusate and dialysate (5 μl each) to 3.0 ml of Ultima Gold scintillation liquid (Packard Instruments, Gronningen, The Netherlands).

The concentration of stable metabolites of NO, nitrate and nitrite (NOx), was measured using fluorometric assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's instructions.

Data acquisition and analysis

Heart rate was obtained from an electrocardiogram, while arterial pressures were monitored with transducers positioned at the level of the heart (Pressure Monitoring Kit, Baxter, Deerfield, IL, USA). Blood gases and haemoglobin concentrations were measured using an ABL725 analyzer (Radiometer, Copenhagen, Denmark). Leg mass was calculated from whole-body dual-energy X-ray absorptiometry scanning (Prodigy, GE Healthcare, Chalfont St Giles, UK). Pulmonary oxygen uptake was measured online (Quark CPET, Cosmed, Italy). Maximal oxygen uptake (Inline graphic) in the young and elderly subjects was determined during an incremental bicycle ergometer exercise test (Excalibur Sport, Lode, The Netherlands) in which the oxygen uptake was determined with a metabolic system (Quark b2 system, Cosmed, Rome, Italy).

Statistical analysis

A two-way repeated measures ANOVA was performed to test significance within and between trials. A two-way ANOVA was used to test significance between the young, elderly and peripheral artery disease subjects within trials. Following a significant F test, pair-wise differences were identified using Tukey's honestly significant difference (HSD) post hoc procedure. The significance level was set at P < 0.05 and data are means ± SEM unless otherwise indicated.

Results

The effect of NOS inhibition on leg haemodynamics at rest and during passive leg movement

Baseline leg blood flow was 0.32 ± 0.03 and increased during passive leg movement to a peak value of 0.92 ± 0.14 litre min−1 (20 s) and remained elevated during the 2 min of passive leg movement (P < 0.05; Fig. 1). The increase in leg blood flow was paralleled by an increase in LVC (P < 0.05) as MAP did not change. The increase in leg O2 delivery was paralleled by a decrease in a-vO2 difference (Table 2; P < 0.05). However, leg Inline graphic was increased by 5–12 ml min−1 during the first 2 min of passive leg movement (P < 0.05).

Figure 1. Leg haemodynamics during baseline conditions and passive leg movement.

Figure 1

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Data are means ± SEM for 7 subjects. *Different from baseline conditions, P < 0.05; #different from control, P < 0.05.

Table 2.

Blood gas variables and Inline graphic during baseline conditions and passive leg movement with and without inhibition of NO formation

Control (s) l-NMMA (s)
Baseline 30 60 90 120 150 180 Baseline 30 60 90 120 150 180
Haemoglobin (g l−1)
 a 146 ± 2 146 ± 2 147 ± 2 147 ± 2 147 ± 2 146 ± 3 148 ± 1 149 ± 3 148 ± 3 148 ± 3 148 ± 3 150 ± 3 151 ± 3 163 ± 3
 v 146 ± 2 147 ± 2 148 ± 2 147 ± 2 147 ± 3 148 ± 3 150 ± 2 148 ± 3 147 ± 3 147 ± 3 147 ± 2 150 ± 3 151 ± 4 152 ± 4
Inline graphic (mmHg)
 a 100 ± 2 96 ± 2 96 ± 2 98 ± 2 98 ± 3 98 ± 3 96 ± 2 105 ± 3 98 ± 1 98 ± 1 97 ± 3 97 ± 3 96 ± 3 97 ± 1
 v 33 ± 1 38 ± 3 40 ± 2 40 ± 2 38.8 ± 3 37 ± 2 39 ± 1 26 ± 1 26 ± 1 28 ± 1 29 ± 2 28 ± 2 27 ± 2 29 ± 2
O2 saturation (%)
 a 97.9 ± 0.2 97.6 ± 0.2 97.6 ± 0.2 97.5 ± 0.3 97.5 ± 0.3 97.7 ± 0.2 97.6 ± 0.1 98.1 ± 0.2 97.8 ± 0.1 97.8 ± 0.1 97.5 ± 0.3 97.4 ± 0.3 97.6 ± 0.3 97.6 ± 0.2
 v 58.1 ± 3.3 70.6 ± 4.1* 70.5 ± 3.9* 70.2 ± 4.5* 67.0 ± 5.2* 65.3 ± 5.0* 69.1 ± 2.9* 40.9 ± 3.8# 42.0 ± 3.0# 46.5 ± 4.2# 46.7 ± 4.1*# 46.1 ± 4.0# 43.6 ± 3.7# 47.3 ± 3.7#
O2 content (ml l−1)
 a 195 ± 3 194 ± 2 195 ± 3 195 ± 3 196 ± 3 195 ± 4 198 ± 2 199 ± 4 197 ± 3 197 ± 3 197 ± 3 199 ± 4 200 ± 4 203 ± 4
 v 115 ± 7 120 ± 9* 142 ± 9* 141 ± 11* 134 ± 12* 132 ± 12* 140 ± 7* 82 ± 7# 84 ± 6# 92 ± 7# 93 ± 8# 94 ± 8*# 90 ± 8# 97 ± 6#
Leg a-vO2 difference (ml l−1)
80 ± 7 54 ± 8* 54 ± 8* 54 ± 8* 60 ± 10* 63 ± 11* 58 ± 6* 117 ± 8# 113 ± 5# 104 ± 9*# 103 ± 8*3 105 ± 5# 110 ± 7# 106 ± 9#
Leg Inline graphic (ml min−1)
24 ± 3 43 ± 3* 31 ± 3* 29 ± 4* 33 ± 5* 32 ± 5 29 ± 1 22 ± 2 23 ± 3# 24 ± 3 22 ± 2 22 ± 3 23 ± 3 25 ± 2
Inline graphic (mmHg)
 a 41 ± 1 42 ± 0 42 ± 0 42 ± 1 42 ± 1 42 ± 1 42 ± 1 41 ± 1 42 ± 1 42 ± 1 41 ± 1 41 ± 1 40 ± 0 40 ± 1
 v 47 ± 2 46 ± 2 49 ± 1 49 ± 2 49 ± 2 50 ± 1 49 ± 2 49 ± 2 49 ± 2 51 ± 1 51 ± 1 51 ± 1 51 ± 1 51 ± 2
pH
 a 7.40 ± 0.01 7.40 ± 0.00 7.40 ± 0.00 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.40 ± 0.01 7.41 ± 0.01 7.41 ± 0.01
 v 7.38 ± 0.01 7.37 ± 0.01 7.37 ± 0.01 7.37 ± 0.01 7.37 ± 0.01 7.36 ± 0.01 7.37 ± 0.01 7.37 ± 0.01 7.38 ± 0.01 7.37 ± 0.01 7.37 ± 0.01 7.36 ± 0.01 7.36 ± 0.01 7.37 ± 0.01
Lactate (mmol l−1)
 a 1.9 ± 0.3 1.8 ± 0.2 1.8 ± 0.2 1.9 ± 0.2 1.9 ± 0.2 1.7 ± 0.2 1.7 ± 0.2 1.6 ± 0.2 1.7 ± 0.2 1.7 ± 0.2 1.6 ± 0.2 1.6 ± 0.2 1.5 ± 0.1 1.5 ± 0.2
 v 1.3 ± 0.1 1.2 ± 0.1 1.4 ± 0.1 1.6 ± 0.2 1.6 ± 0.3 1.6 ± 0.2 1.8 ± 0.2 1.4 ± 0.1 1.3 ± 0.2 1.5 ± 0.2 1.5 ± 0.1 1.7 ± 0.2 1.6 ± 0.1 1.8 ± 0.2
Glucose (mmol l−1)
 a 5.5 ± 0.2 5.6 ± 0.3 5.6 ± 0.3 5.6 ± 0.3 5.6 ± 0.3 5.7 ± 0.3 5.7 ± 0.3 5.6 ± 0.3 5.6 ± 0.2 5.6 ± 0.2 5.6 ± 0.2 5.6 ± 0.2 5.4 ± 0.1 5.3 ± 0.0
 v 5.1 ± 0.3 4.7 ± 0.3 5.1 ± 0.3 5.1 ± 0.2 5.1 ± 0.3 5.0 ± 0.2 5.0 ± 0.3 4.7 ± 0.2 4.8 ± 0.3 4.9 ± 0.2 4.9 ± 0.2 4.8 ± 0.2 4.8 ± 0.2 4.8 ± 0.2
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Data are mean ± SEM for 7 subjects. *different from baseline conditions, P < 0.05; #different from control, P < 0.05.

During NOS inhibition, baseline leg blood flow, vascular conductance and O2 delivery were lower compared to control conditions (P < 0.05), whereas the a-vO2 difference was increased (P < 0.05) and MAP and leg Inline graphic were similar. During passive leg movement, leg blood flow and vascular conductance increased at 20 s, but then returned to baseline values, whereas leg a-vO2 difference and leg Inline graphic did not increase compared to baseline. MAP increased at 60, 120, 150 and 210 s (P < 0.05). Accounting for the change in baseline leg blood flow with NOS inhibition, leg blood flow and vascular conductance were lower during passive leg movement in the presence of NOS inhibition compared to control (P < 0.05). Passive exercise did not alter heart rate in either the control or the NOS inhibition trials and there was no difference between the two trials.

Venous plasma and muscle interstitial NOx levels during passive leg movement

Passive leg movement increased venous NOx levels from 35 ± 5 to 62 ± 11 μmol l−1 (P < 0.05; Fig. 2), whereas NOx levels remained unchanged in the muscle interstitium during passive leg movement.

Figure 2. Femoral venous plasma and muscle interstitial NOx concentrations during baseline conditions and passive leg movement.

Figure 2

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Data are means ± SEM for 6 subjects. *Significantly different from baseline, P < 0.05.

Hyperaemic response to passive leg movement and arterial ACh infusion

Leg blood flow and vascular conductance (AUC) during passive leg movement was correlated to leg blood flow (r2 = 0.570, P = 0.005) and vascular conductance (r2 = 0.704, P < 0.001) during arterial ACh infusion (Fig. 3).

Figure 3. Leg blood flow (upper panel) and vascular conductance (lower panel) during passive leg movement in 20- to 70-year-old individuals plotted against the same variables during ACh infusion.

Figure 3

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Data are means ± SEM for 12 subjects.

Hyperaemic response to passive leg movement in elderly and patients with peripheral artery disease

Baseline leg blood flow was lower in the elderly and peripheral artery disease subjects (0.22 ± 0.05 and 0.13 ± 0.03 litre min−1, respectively), compared to the young subjects (P < 0.05). Passive leg movement increased leg blood flow in the elderly subjects at 20, 30 and 40 s (P < 0.05) where after leg blood flow returned to baseline levels. Leg blood flow was lower throughout passive leg movement in the elderly compared to the young subjects (P < 0.05; Fig. 4). Passive leg movement did not increase leg blood flow in the patients with peripheral artery disease (Fig. 5). Compared to the young subjects, leg blood flow (AUC) was lower before and during passive leg movement in both groups (P < 0.05).

Figure 4. Leg blood flow during baseline conditions and passive leg movement in young and elderly subjects.

Figure 4

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Data are means ± SEM for 14 subjects. *Different from baseline conditions, P < 0.05; #different from young, P < 0.05.

Figure 5. Leg hyperaemia induced by passive leg movement in healthy individuals and individuals with peripheral arterial disease (PAD).

Figure 5

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Leg blood flow and the change in leg blood flow from baseline to passive leg movement in young and elderly healthy subjects and patients with peripheral artery disease (left). Individual leg blood flow response and the changes in leg blood flow expressed in relation to age (right). Area under the curve (AUC) was calculated from continuous Doppler tracings during 90 s of passive leg movement. Displayed correlations are for healthy subjects. *Significantly different from baseline, P < 0.05; #different from young subjects, P < 0.05.

Discussion

The main findings of this study were that: (1) inhibition of NO synthesis lowered the vasodilatory response to passive leg movement by ∼90%, (2) passive leg movement increased venous plasma NOx levels, whereas interstitial NOx levels remained unchanged, (3) the hyperaemic response to passive leg movement was correlated to the vasodilatory response to ACh, (4) leg blood flow increased in the healthy elderly subjects, but the increase was lower compared to the young subjects, and (5) leg blood flow did not increase with passive exercise in subjects with peripheral artery disease. These results suggest that the hyperaemic response to passive leg movement is dependent on NO and that the source is likely to be the endothelium. Passive leg movement can therefore be used to test endothelial NO function in the leg.

The present study demonstrates that inhibition of NO formation reduces the hyperaemia observed during passive leg movement in young, healthy individuals by ∼90%. NO formation can be stimulated both chemically and mechanically and given that passive leg movement involves no alteration in muscle activation (Høier et al. 2010) and only a small increase in metabolism, it may be assumed that the enhanced NO formation with passive movement mainly occurs via mechanical stimuli. l-NMMA is likely to inhibit both nNOS and eNOS, but the increase in venous NOx and not muscle interstitial fluid during passive movement suggest that the source of NO is likely to be eNOS in the endothelium and not nNOS in the muscle tissue. Passive movement leads to alterations in both shear stress and passive stretch of the tissue (Cheng et al. 2009), both of which have been found to stimulate NO formation in vitro (Pohl et al. 1986; Joannides et al. 1995; Kooijman et al. 2008). In contrast, experimental compression of endothelial cells in vitro does not appear to have an effect on NO release (Dai et al. 2002). Collectively, these observations suggest that the enhanced NO formation during passive movement is likely to be induced by either shear stress or passive stretch or potentially both factors combined. It is noteworthy that NOS blockade almost completely abolished the flow response to passive movement. Leg blood flow was only increased after 20 s of passive leg movement during NOS inhibition, which is consistent with a slower endothelial response to shear stress (Pyke et al. 2004; Shipley et al. 2005). Despite this initial NO-independent increase in leg blood flow, the overall hyperaemic response was 90% lower during the first 90 s of passive leg movement, suggesting that NO plays an important role in the hyperaemic response to passive leg movement. This was further supported by the correlation between the hyperaemia during passive leg movement and infusion of the endothelium dependent vasodilator ACh (Furchgott & Zawadzki, 1980). The 90% lower hyperaemic response when NO formation was inhibited also refute the hypothesis that a central response to passive leg movement is an important component of the hyperaemic response (Trinity et al. 2010).

To gain further insight into the role of the endothelium in the hyperaemic response to passive leg movement, we compared the young individuals to two populations known to have reduced endothelial function. When passive leg movement was performed in the elderly subjects, the hyperaemic response was attenuated (McDaniel et al. 2010), which is in agreement with a reduced endothelial function with advancing ageing (Taddei et al. 1995). We did not measure or test NO function or test endothelium-independent vasodilatation in these individuals and therefore it cannot be excluded that the lower hyperaemic response in the elderly subjects was associated with other mechanisms. However, ageing appears not to affect endothelium-independent vasodilatation (Celermajer et al. 1994; Kirby et al. 2010), suggesting that the lower hyperaemic response was not caused by a reduced response to NO. Leg blood flow did not increase during passive leg movement in elderly individuals with peripheral artery disease, which is a disease known to be associated with severely reduced NO function (Böger et al. 1997). The observations in healthy elderly and elderly with peripheral artery disease therefore appear to support our observations in healthy young men, suggesting that the hyperaemic response to passive leg movement is NO dependent.

In contrast to the present findings during passive leg movement, NOS inhibition does not alter exercise hyperaemia in the leg (Rådegran & Saltin, 1999; Frandsen et al. 2001) and only up to ∼35% in combination with pharmacological inhibition of other vasodilator systems (Boushel et al. 2002; Mortensen et al. 2007,2009b). This discrepancy is likely to be a reflection of the mechanisms that mediate hyperaemia under the different conditions. During exercise, blood flow appears to be regulated by an interaction between several vasodilator systems, including NO, which ensures adequate O2 delivery even when one system is impaired. In contrast, the hyperaemia in response to passive movement occurs without need for an increased oxygen supply and is simply the result of a mechanical signal. Interestingly, the small (∼20 ml min−1) increase in leg Inline graphic during passive leg movement (González-Alonso et al. 2008; Høier et al. 2010) was abolished during NOS inhibition. A direct effect of NO on mitochondrial respiration does not appear to explain this observation, because leg Inline graphic was not altered during baseline conditions. Instead, mechanical stress and/or the increase tissue O2 delivery may have caused a local increase in metabolism. In support of the latter, we have observed a similar tendency during arterial ATP infusion (Mortensen et al. 2009a).

Implications

Flow mediated dilatation (FMD) in the brachial artery is a widely used method to evaluate endothelial function in patients (Celermajer et al. 1992; Thijssen et al. 2011a), and although FMD is commonly interpreted to reflect eNOS function, other vasodilators contribute to the hyperaemic response following occlusion of the artery (Tschakovsky & Pyke, 2005). Atherosclerosis and peripheral artery disease can specifically affect one leg or be more pronounced in the leg compared to the arm (Fukudome et al. 1997; Tendera et al. 2011) and it is likely that there are differences in endothelium-dependent and endothelium-independent vasodilatation between the arms and legs (Newcomer et al. 2004) due to large differences in hydrostatic pressure (Rowell, 1993). Furthermore, the larger volume of the legs may provide a better estimate of overall cardiovascular function than the forearm (Thijssen et al. 2011b). The findings that passive leg movement is NO dependent and the hyperaemic response is correlated to the response to ACh suggest that passive leg movement can be used to specifically test endothelial NO function of the legs.

In conclusion, the present results demonstrate that the hyperaemic response to passive leg movement is NO dependent and that the source of NO is likely to be the endothelium. Although the precise mechanisms remain unclear, it is suggested that mechanical forces such as shear stress and passive stretch cause endothelial release of NO. Furthermore, the hyperaemic response is attenuated in elderly and abolished in patients with peripheral artery disease. The hyperaemic response is also correlated to the vasodilatation induced by ACh. Passive leg movement could therefore be a simple, non-invasive method to specifically evaluate NO function in humans.

Acknowledgments

This study was supported by a grant from the Lundbeck foundation, the Danish Council for Independent Research – Medical Sciences and from the University of the Sunshine Coast. S.P.M. was supported by a grant from the Danish Council for Independent Research – Medical Sciences. M.N. was supported by a grant from the Lundbeck foundation. C.A. is a member of the National Health and Medical Research Council funded National Centre of Research Excellence for Peripheral Arterial Disease (NCRE-PAD, Australia).

Translational perspective

Endothelial dysfunction plays a key role in the pathogenesis of micro- and macrovascular complications observed in pathological conditions such as diabetes, atherosclerosis, hypertension and peripheral artery disease, and it is a risk factor for cardiovascular events. The findings that the hyperaemia during passive leg movements is mediated by NO and correlated to ACh induced vasodilatation suggest that passive leg movement can be used to test endothelial function. A test for endothelial function in the leg is important because there are differences in vascular function between arms and legs and some disease states only affect the legs. In addition, the >10-fold larger volume of the leg compared to the arm is more likely to reflect the vascular function of the cardiovascular system. In a standardized set-up, passive leg movement can therefore be a simple, non-invasive tool to evaluate endothelial NO function of the lower limb.

Glossary

l-NMMA

NG-mono-methyl-l-arginine

LVC

leg vascular conductance

NO

nitric oxide

NOx

nitrate/nitrite

Inline graphic

oxygen uptake

Author contributions

The contributions of the authors were as follows. Conception and design of the study: S.P.M, M.N., C.D.A, M.W. and Y.H.; collection, analysis, and interpretation of data: S.P.M, M.N. C.D.A, M.W. and Y.H.; drafting the article or revising it critically for important intellectual content: S.P.M, M.N., C.D.A, M.W. and Y.H. All authors approved the final version.

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